Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus and the causative infectious agent of both HTLV-1- associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic inflammatory disease of the central nervous system, and adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal disease of CD4+ T cells. An ideal therapeutic strategy against HAM/TSP is still not established and once neurological symptoms begin, they progress quickly leading to deterioration in quality of life. ATL is chemotherapy-resistant with a median survival time of <1 year. The lack of effective therapies suggests an imminent need for new approaches to treatment or prevention. We propose a novel approach utilizing the CRISPR bacterial immunity system to disable or disrupt the HTLV-1 proviral genome and cell proliferative and ultimately tumorigenic effect on T cells. The CRISPR system includes the Cas9 endonuclease that is targeted by a guide RNA (gRNA) to introduce a specific double strand break (DSB) into genomic DNA. These DSBs are largely repaired by the error-prone non-homologous end- joining (NHEJ) pathway in most human cells, which typically introduces insertions and deletions (indels) at the repair junction. Indels may alter the reading frame of genes or disrupt essential regulatory elements or RNA stem-loop structures. However, the broad effects of using the CRISPR system to target the HTLV-1 genome are unknown. This proposal contains two highly focused Specific Aims: 1.) Utilize CRISPR/Cas9 technology to disable the HTLV-1 provirus and its proliferative inducing and tumorigenic effects on infected T cells, and 2.) To determine the therapeutic efficacy of CRISPR/Cas9-based genome editing against HTLV-1 NOG SCID transplant mouse model. We will generate a library of CRISPR gRNAs targeting key regions of the HTLV-1 genome, including the long terminal repeats (LTRs), the tax gene, and the hbz gene. This library will be characterized for viral infectivity, transcription, cellular proliferation, and off-target editing. The nature and location of mutations that effectively disable HTLV-1 will be analyzed by sequencing. We will further determine if the CRISPR induced mutations prevent HTLV-1 cell growth and tumor induction in vivo. Ultimately, this exploratory grant will provide a quantitative and adaptable platform for probing the efficiency of CRISPR editing to disable HTLV-1 and prevent or disrupt viral gene expression, infected cell growth and ultimately host neurological/immunological complications or oncogenesis. These data will yield significant insights into the suitability and practicality of genome editing technologies targeting HTLV-1.